Part:BBa_K5124028
Cas13a PLAUR_target for transcription
Usage and Biology
The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.
In 2021 McLoughlin et al. published RNA-Seq data from cattle infected with bTB at several timepoints during the disease progression [1]. They identified 19 potential biomarkers that were present across the entire length of the infection time course. We would have liked to have tested all 19 sequences but after two rounds of the design-build-test-learn cycle (see our Wiki) we focused on:
CXCL8- chemokine ligand 8, involved in infection response and tissue injury.
FOSB- FBJ murine osteosarcoma viral oncogene homologue B, plays a role in regulating cell proliferation, differentiation and transformation.
NR4A1- nuclear receptor subfamily 4, group A, member 1, plays a role in inflammation and apoptosis.
PLAUR- plasminogen activator, urokinase receptor, a biomarker of inflammation.
RGS16- regulator of G-protein signalling 16, linked to several disease states.
This composite part contains a 5’ T7 promoter and a target sequence; a 150-nucleotide RNA sequence from the Bos taurus genome (BioProject accession number PRJNA450837). Once transcribed this sequence contains a 24-nucleotide sequence that is complimentary to the spacer sequence PLAUR (see basic part BBa_K5124022 for details). This target sequence was used in our Cas13a assays as a mimic of bTB infected samples from cattle. In the final detection system, this part could be used as a positive control.
Design and Characterisation
This sequence was synthesised by IDT as a composite part containing a 5’ T7 promoter (BBa_K5124041) and Type IIs compatible prefix and suffixes. As we needed the RNA sequence be identical to the Bos taurus genome, we were unable to remove the PstI restriction enzyme site. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.
The respective sgRNA is BBa_K5124039
Results
Results from in-vitro transcription reactions are shown below.
All columns on the RNA tape showed there was RNA present around the expected length for sgRNA, and target RNA.
Due to the small size of the target RNA sequence and the upper marker in the sample buffer being degraded, the length of RNA sequences will not be accurate. A 153-target nucleotide peak was expected, the peak at 70 nucleotides demonstrates that a short RNA was transcribed. We were happy that this demonstrates successful transcription of our RNA. A peak at 119 nucleotides may indicate that some template DNA was not cleaved (see protocol on Wiki) and transcription carried on past the end of the target RNA sequence. There is also a large board peak, above 2000 bp, which is likely from environmental contamination. The concentration of PLAUR target RNA was determined to be 149.6 ng/ul which was a usable concentration for our final tests.
Results from our final tests are shown below.
We mixed our Cas13a, an sgRNA, the corresponding target RNA and our fluorescent probes and saw an increase in fluorescence with time for PLAUR, CXCL8, RGS16 and NR4A1. Tis shows that when activated by the correct sgRNA Cas13a binds to the target and cleaves the probe. However, there was no increase in fluorescence for FOSB. This is because we discovered a mistake in the folding of the FOSB sgRNA. However this proved beneficial as it shows that if the sgRNA does not fold correctly Cas13a cannot cleave the probes.
Conclusion
We successfully expressed our Cas13a enzyme and achieved reasonable purity. We can tell the Cas13a system worked because we got positive results when the Cas13a, sgRNA, target and probe were added together (as shown in the graph above), but also because we had negative controls that caused no reaction. These were:
The system missing one of the 4 parts showing all parts are necessary.
The system with the wrong target showing that the system only activates with the correct RNA sequence (data not shown on this graph please see Measurement section on Wiki).
The system with the spacer FOSB which folded incorrectly, showing the testing for the folding of sequences was necessary.
References
1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.
2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.
3. Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 136
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 136
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 136
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 136
- 1000COMPATIBLE WITH RFC[1000]
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